M2 P7- Cellular Neurobiology And Development - 2009

M2 P7- Cellular Neurobiology and Development - 2009 Lundi 21/9

Mardi 22/9

Mercredi 23/9

Jeudi 24/9

Vendredi 25/9

9h-11h Frédéric Saudou, François Tronche, Thierryy Galli,,[email protected], @ , Jean-Christophe p Poncer,, Isabelle Caillé [email protected] Caillé, francois tronche@gmail co [email protected], CoursMécanismes moléculaires [email protected]@snv.jussieu. m, Approches de génétique Optionnel d'Introduction:de la neurodégénation dans moulin.inserm.fr, Plasticité fr, Traduction locale dansmoléculaire pour l'étude des Rappels de M1 / Optionalla maladie de Huntington / synaptique dans les les neurones / Neuronalfonctions cérébrales / Introductory Lecture fromMolecular mechanisms of réseaux corticaux / Synaptic local translation Molecular Genetics for the M1§ plasticity in cortical networks neurodegeneration in study of brain functions H ti t ' disease Huntington's di 11h1513h15

14h1514h15 16h15

Evelyne Bloch-Gallego, blochJamel Chelly, [email protected] ,[email protected], Facteurs de guidage ettitre Causes génétiques etIsabelle Caillé, Nathalie Spassky, concepts [email protected] Thierry Galli,réorganisations [email protected]. [email protected], Biologieintracellulaires durant laimpliqués dans le déficitr, Les cellules gliales Cellules ciliées et cellulaire du Neurone / Cellcoissance axonale et lamental / Genetic causescomme cellules souches neurogénèse/ Ciliated cells migration neuronale neurobiologicalneurales / Glial cells as biology of the neuron /and and neurogenesis Guiding factors andconcepts involved in mentalneural stem cells intracellular rearrangementsdeficiency (learning during axonal outgrowthdisability) and neuronal migration

Alessandra Pierani, [email protected], Régionalisation dorsoLydia Danglot, Nathalie Rouach, Thierry Galli, ventrale du tube neural: [email protected], nathalie.rouach@[email protected], Trafic domaines p Développement pp et progéniteurs g et france fr france.fr, Astrocytes et membranaire et synaptogenèse de développement des classesExamen Final / Final Exam plasticité synaptique / différenciation neuronale / l'hippocampe / Development neuronales / Dorso-ventral Astrocytes and synaptic Membrane Traffic and and synaptogenesis of the regionalisation of the neural plasticity neuronal differentiation hippocampus tube : progenitor domains and development of neuronall classes l

§ Ce cours a été demandé par les étudiants des années précédentes. Il est recommandé pour ceux qui n'ont pas suivi le cours de M1 d'I Caillé et T Galli. This lecture was requested by the students of last year. It is intended for the students who did not follow the M1 course by I Caillé and T Galli.

Connect… • http://sites.google.com/site/insermu950/ home • [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], [email protected]

Introduction • • • •

How the nervous system is organized Nerve cell types and roles Excitability and electrical signals Graded and action potentials initiation and conduction • Neurotransmitters and signal conduction cell o ce cell to • Modulation and integration of the signals

Organization g of the Nervous System y • Rapid communication for homeostatic balance • Emergent properties of intelligence & emotion • Central Nervous system y ((CNS)) • Peripheral Nervous system (PNS)

Organization g of the Nervous System y

Figure 8-1: Organization of the nervous system

A Typical yp Neuron Overview

• • • •

Dentrites Cell Body Axon Terminal

Figure 8-2: Model neuron

Diverse Neuron Forms and Functions

• • • • •

Pseudounipolar Bipolar Anaxionic M lti l CNS Multipolar–CNS Multipolar–efferent p

Diverse Neuron Forms and Functions

Figure 8-3: Anatomic and functional categories of neurons

Metabolism and Synthesis in a Neuron

• Cell body site of energy generation and synthesis • Axonal transport – Vesicles – • Fast axonal transport to terminal • Retrograde to cell body

– Electrical depolarizations

Metabolism and Synthesis in a Neuron

Figure 8-4: Axonal transport of membranous organelles

Glial Cell Functions

• Support neuron bodies, form myelin sheaths • Barriers B i b between t compartments t t • Scavenger/defense & metabolic assistance

Neuron & friends

Glial Cell Functions

Figure 8-5: Glial cells and their functions

Electrical Signals: Ionic Concentrations and Potentials • Nernst & GHK Equations predict • Membrane potential • Cell concentration gradients • [Na+, Cl- & Ca2+] higher in ECF • [K+] higher ICF • Depolarization causes electrical signal • Gated channels control permeability

Electrical Signals: Ionic Concentrations and Potentials

Table 8 8-2: 2: Ion Concentrations and Equilibrium Potentials

Graded Potentials • Incoming signals – Vary in strength – Lose strength over distance – Are slower than action potentials (AP)

• Travels to trigger zone – Subthreshold – • Too weak • No generation of AP

– Suprathreshold – generate AP

Graded Potentials

Figure 8-7: Graded potentials decrease in strength as they spread out from the point of origin

Trigger Zone: Cell Integration and I iti ti off AP Initiation

• Excitatoryy signal: g depolarizes, p reduces threshold • Inhibitory signal: hyperpolarizes, hyperpolarizes increases threshold

Trigger Zone: Cell Integration and I iti ti off AP Initiation

Figure 8-8a: Subthreshold and suprathreshold graded potentials in a neuron

Trigger Zone: Cell Integration and I iti ti off AP Initiation

Figure 8-8b: Subthreshold and suprathreshold graded potentials in a neuron

Action Potential Stages: Overview

• "All All or none" none • Signal does not diminish over distance

Action Potential Stages: Overview

Figure 8-9: The action potential

Membrane & Channel Changes during an Action Potential

• • • •

Initiation Depolarization Signal g p peak Repolarization

Membrane & Channel Changes during an Action Potential

Figure 8-10: Model of the voltage-gated channel Na+

Introduction to Cell Biology of the Neuron

2 compartments: compartments: o • Axon • Somatodendritic

Axon (NF)

Spinal cord neuron Soma + Dendrites (MAP 2) Hippocampal neuron

Neuron Parts: Major M j sites it to receive input

Major sites for output

The q question we are focusing g on: Neuron polarization: Development of axon & dendrites.

Neuronal polarity: p y domains

NEURONS ARE POLARIZED

LDLR

L1-CAM

Different axonal domains

CamKII

Pioneering in vitro study by Ganry Banker i 1980’s-1990’s in 1980’ 1990’

Craig AM, Banker G. Annu Rev Neurosci. 1994;17:267 310. 1994;17:267-310.

Thus, the question of neuron polarization can be simplified as the selection of one neurite growth to become axon during the transition from stage 2 to 3 in the in vitro case of this specific type of neuron.

Neuronal Differentiation

Molecular markers for axon or dendrite Tau1 au Axon: Tau1 Tau1, GAP43, GAP43 synapsin synapsin, synaptotagmin …

Dendrite: MAP2, Glycine receptor, GABAa receptor …

MAP2

Hippocampal Neuron Polarization from Stage 2 to Stage 3

da Silva JS, Dotti CG. Nat Rev Neurosci. 2002 Sep;3(9):694-704.

Neuron growth cone

Evident from intensive axon guidance researches, neurite growth g y controlled by y the structure in its tip p ---growth g cone. is tightly Maybe the question of polarization can be furthur simplified as the selection of growth cone for growth.

Neuronal differentiation I. Membrane growth and polarized sorting Precursor

Immature Neuron

Mature Neuron

Dendrites: TfR and Golgi g

Axon: Synaptotagmin

Neuronal differentiation

II. Plasticity in membrane growth Precursor

P-lysine

Immature Neuron

Mature Neuron

L1

Neuronal differentiation III. Directed g growth th

From: Hong-jun Song and Mu-ming Poo Nature Cell Biology 2001

1. Role of membrane trafficking in neuritogenesis

Phase contrast time-lapse recording of a neuron forming an axon. The video shows a rapid p p playback y of a 16 hour recording g with one image taken every 10 minutes.

Membrane traffic: basic mechanisms

Synaptobrevin 2

Regulation: Rab GTPases

Syntaxin 1 SNAP25 Brunger, 1998

Cai et al, Dev Cell 2007

Neuron maturation and membrane trafficking Neurite: Kinesin-dependent transport of cytoskeleton components & regulators, and of vesicle-associated cargos g Arimura & Kaibuchi Nat Neurosci 2007

Growth cone: Main M i site i off membrane insertion Craig 1995, & others

SNAREs at the plasma membrane EFFECT ON NEURON MATURATION

NO EFFECT ON NEURON MATURATION

•

•

SNAP-25 Genetic invalidation in mouse

•

SNAP-23? Could compensate SNAP-25 absence

Washbourne Nat Neurosci 2002

W hb Washbourne N Natt N Neuroscii 2002

Syntaxin 1 Genetic invalidation in worm

Syntaxin 3 RNA interference

Saifee Mol Biol Cell1998

•

Genetic invalidation in mouse

•

RNA interference

Fujiwara J Neurosci 2006 Darios & Davletov Nature 2006

•

Darios & Davletov Nature 2006

On the vesicular side TeNT cleavage of Synaptobrevin 2 TeNT

Synaptobrevin 2 KO mic

Syb 2

Contrast

FM1-43 uptake

Osen-Sand, J. Comp. Neurol. 1996

Schoch, Science 2001

Neuritogenesis is TeNT resistant Precursor

Immature Neuron

Mature Neuron

Growth cone

Tetanus Neurotoxin resistant Syb2 independent

TI-VAMP in neuritogenesis

Suivie S i i d de GFP GFP-TIVAMP TIVAMP pendant la neuritogenèse

TI-VAMP in growth cones

TI-VAMP Syb/VAMP2

(Coco et al., J.Neurosci.1999)

Ursula Schenk

Presynaptic markers and growth th cone proteins t i are enriched in GCP preparations

TI-VAMP is essential for neurite outgrowth in neurons

Alberts & al MBoC 2003

Flux of secretory vesicles in the growing neurite: a main player

Krasimira Tsaneva-Atanasova David Holcman

TI VAMP RFP TI-VAMP Synaptobrevin 2 GFP

What is needed to build a neurite?

• Vesicles: TI-VAMP mediated transport and regulators

• Microtubules and regulators

• Actin filaments and regulators

Involvement of syntaxin y 3 in neurite outgrowth

Darios & Davletov, Nature 2006

Neurite outgrowth is impaired in SCG explants from Syt VII-/-mice

Rao & al JBC 2004 Arantes & al JJ. Neurosci Neurosci. 2006 Copyright ©2006 Society for Neuroscience

Role of exocytosis in neuronal morphogenesis Basic molecular mechanism mediated by: • TI-VAMP/VAMP7 as v-SNARE • Syntaxin S 3 as t-SNARE S • Synaptotagmin VII TIVAMP

TI-VAMP/VAMP7 Syntaxin3

Cargo: Neural cell recognition molecule l l L1 -L1: L1: member of the immunoglobulin superfamily of cell adhesion dh i molecules l l -involved in axonal growth and pathfinding -mutation in man: MASAsyndrome y ((mental retardation, spasticity, hydrocephalus) -KO-mice: KO i malformation lf i off the corticospinal tract

F. Rathjen http://www.mdc-berlin.de/~devneuro/fgr-intr.htm

L1-CAM is a TI-VAMP’s Cargo g

Alberts & al MBoC 2003

TI-VAMP: v-SNARE mediating neurite outgrowth Targeting g g

TI-VAMP Syb/VAMP2

AP-3 Auto inhibition Auto-inhibition

LONGIN 1

SNARE TM 180

120

TI-VAMP TI VAMP

220

(Coco et al., J.Neurosci.1999)

SNARE TM

Neurite outgrowth Longin-TIVAMP



Longin-TIVAMP



ARNi



Synaptobrevin 2

TeNT

Conclusion: Integration of signaling, actin, and exocytosis in neurite outgrowth -TI-VAMP is a vesicular SNARE that is necessary for neurite outgrowth -TI-VAMP transports the IgCAM L1 L1. The TI-VAMP dependent membrane trafficking regulates the stability of L1-dependent adhesive contacts -L1 mediated adhesion induces a polarization of TI-VAMP vesicles to sites of contact -The exocytosis of TI-VAMP is positively controlled actin dynamics and cdc42

cdc42

cdc42

cdc42

Philipp Alberts

2. The cytoskeleton and neuronal polarity

MICROTUBULES (organising centres and polarity)

+

Basal body

Cillia or Flagella

MTOC Centrosome Centrioles

Migrating cell + + +

+

Spindle poles

Neurones Mitotic Mit ti spindle i dl Dividing cell

+

BBA, 1376:27 (1998)

Cell C ll polarization l i ti requires i capture t off microtubules at the leading g edge g

Role of microtubules and their regulators

Motors carrying different g cargoes in different directions

fibroblast

neuron

Anterograde and retrograde transport

Cargo structures

Overall rate (pulse labeling) 200–400 mm/da (2– 5 µm/s)

Instantaneous rate (light microscopy)

Directionality

Duty ratio

1–5 µm/sb

Anterograde

High

Endocytic vesicles, lysosomes, autophagosomes p g (fast retrograde)

100–250 mm/da (1– 3µ µm/s))

1–3 µm/sb

Retrograde

High

Mitochondria

<70 mm/dc (<0.8 µm/s)

0.3–0.7 µm/sd

Bidirectional

Intermediate

Microfilaments, Mi fil t cytosolic protein complexes (slow component b)

2–8 2 8 mm/d /de (0.02–0.09 µm/s)

U k Unknown

U k Unknown

U k Unknown

Microtubules, neurofilaments (slow component a)

0.2–1 mm/de (0.002– 0.01 µm/s)

0.3–1 µm/sf

Bidirectional

Low

Golgi-derived vesicles (fast anterograde)

Role of kinesins

Centrosome localization determines neuronal polarity

Froylan Calderon de Anda, Giulia Pollarolo, Jorge Santos Da Silva Silva, Paola G G. Camoletto Camoletto, Fabian Feiguin & Carlos G G. Dotti Nature (2005)

Centrosome localization determines neuronal polarity

Froylan Calderon de Anda, Giulia Pollarolo, Jorge Santos Da Silva Silva, Paola G G. Camoletto Camoletto, Fabian Feiguin & Carlos G G. Dotti Nature (2005)

Microtubule stabilization by CRMP2 In cultured hippocampal neurons, one axon and several dendrites differentiate from a common immature process. Here we found that CRMP-2/TOAD-64/Ulip2/DRP-2 (refs. 2-4) level was higher in growing axons of cultured hippocampal neurons, that overexpression of CRMP-2 in the cells led to the formation of supernumerary axons and that expression of truncated CRMP-2 mutants suppressed the formation of primary axon in a dominantnegative manner. Thus, CRMP-2 seems to be critical in axon induction in hippocampal neurons, thereby establishing and maintaining neuronal polarity.

Red: actin

Green: microtubule

Inagaki et al., Nat Neurosci. 2001 Aug;4(8):781-2.

Conversion of p preexisting g dendrite to axon by GSK-3 inhibition

Model

MICROFILAMENTS ACTIN STRUCTURES IN CELLS:

MICROVILLI

STRESS FIBRES FOCAL ADHESIONS

LAMELLIPODIA FILOPODIA (or MICROSPIKES

CONTRACTILE RING (cell division)

HPC neuron, 24h

DNA- blue; µtubules- green; actin- red

HPC neuron, 3 weeks

?

Actin instability in growth cone Local perfusion of cytochalasin D onto a growth cone induces it to grow as an axon, indicating that actin destabilization is sufficient for axon formation. These data strengthen the proposed hypothesis that polarized actin-filament instability determines initial neuronal polarization

Red: actin Green: microtubule

Bradke F, Dotti CG. Science. 1999 Mar 19;283(5409):1931-4.

-The Role of Local Actin Instability in Axon Formation. Frank Bradke and Carlos G. Dotti. (1999) Science 283: 1931-1934 -Establishment of neuronal polarity: lessons from cultured hippocampal neurons, Frank Bradke and Carlos G Dotti (2000). Curr Opin. Neurobiol. 10: 574 581 574-581

Actin instability

Role of the actin cytoskeleton

The sequential activity of the GTPases Rap1B and Cdc42 determines neuronall polarity. l it

Phalloidine Cdc 42 Rap1b

Phalloidine

Schwamborn JC, Puschel AW, Nat Neurosci. (2004) .

The sequential activity of the GTPases Rap1B and Cdc42 determines neuronal polarity. Nat Neurosci. 2004 Schwamborn JC, Puschel AW The establishment of a polarized morphology is an essential step in the differentiation of neurons with a single i l axon and d multiple lti l dendrites. d d it I cultured In lt d ratt hippocampal neurons, one of several initially indistinguishable neurites is selected to become the axon. Both phosphatidylinositol 3,4,5-trisphosphate and d the th evolutionarily l ti il conserved d Par P complex l (comprising Par3, Par6 and an atypical PKC (aPKC) such as PKClambda or PKCzeta) are involved in axon specification. However, the initial signals that establish t bli h cellular ll l asymmetry t and d the th pathways th th t that subsequently translate it into structural changes remain to be elucidated. Here we show that localization of the GTPase Rap1B to the tip of a single i l neurite it is i a decisive d i i step t i determining in d t i i which neurite becomes the axon. Using GTPase mutants and RNA interference, we found that Rap1B is necessary and sufficient to initiate the d development l t off axons upstream t off Cdc42 Cd 42 and d the th Par complex.

The sequential y of the activity GTPases Rap1B and Cdc42 determines neuronall polarity. l it Nat Neurosci. 2004 Schwamborn JC, JC Puschel AW.

Neuronal Polarity: PI3Kinase etc…

Rho GTPases

4. Inside 4 Insideout OR Outside in Outside-in ?

Polarity

Inside-Out Selective sorting of proteins

Inside-Out: sorting signals i l

Inside-Out : signals and sorters

Example of signal: the axonal initial g (AIS) ( ) of Dargent g & coll. segment

Selective sorting or selective retrieval?

Transcytosis of NgCAM in neurons

Raft: axonal signals?

Missorting of the axonal Thy-1 but not of a dendritic membrane protein occurred in sphingolipid-deprived cells. These results indicate that neurons sort a subset of axolemmal proteins by a mechanism that requires the formation of protein-lipid rafts. The involvement of rafts in axonal membrane sorting may explain the neurological deficits observed in patients with certain types of Niemann-Pick disease.

Ledesma Maria Dolores et al. Ledesma, al (1998) Proc Proc. Natl. Natl Acad. Acad Sci Sci. USA 95, 95 3966-3971 3966 3971

Copyright ©1998 by the National Academy of Sciences

Outside-In

Cell polarity is regulated by signaling molecules that localize to the leading edge 1 Membrane receptors (GPCRs 1. (GPCRs, RTKs) detect an asymmetric signal from outside the cell 2 Receptors acti 2. activate ate Ras Ras-like like small G proteins (Rho proteins) 3. Rho proteins induce cytoskeletal changes at the leading (Rac, Cdc42) and trailing (Rho) edges of the cell

IGF1R?

IGF-1 receptor is essential for the establishment of hippocampal neuronal polarity Lucas Sosa, Sebastian Dupraz, Lisandro Laurino, Flavia Bollati, Mariano Bisbal, Alfredo Cáceres, Karl H Pfenninger & Santiago Quiroga Nature Neuroscience 2006

Rho GTPases

Signalling & outgrowth

Growth, Guidance, Synapse… The END

Synaptic transmission: communication between neurons

Two principal kinds of synapses: electrical and chemical

Chemical synapses: the predominant means of communication between neurons

Presynaptic Active Zone

An early experiment to support the neurotransmitter hypothe

Criteria that define a neurotransmitter: 1. Must be p present at p presynaptic y p terminal 2. Must be released by depolarization, Ca++-dependent 3. Specific receptors must be present

Neurotransmitters may be either small molecules or peptide Mechanisms and sites of synthesis are different Smallll molecule S l l transmitters are synthesized at terminals, packaged into small clear-core vesicles (often referred to as synaptic vesicles’ vesicles ‘synaptic

Peptides, or Peptides neuropeptides are synthesized in the endoplasmic d l i reticulum and transported to the synapse, sometimes they are p processed along the way. Neuropeptides are packaged in large dense-core vesicles

Neurotransmitter is released in discrete packages, or quanta

Failure analysis reveals that neurons release many quanta of neurotransmitter when stimulated, stimulated that all contribute to the response

Quantal content: The number of quanta released q by stimulation of the neuron

Quantal Q t l size: i How size of the individual quanta

Quanta correspond to release of individual synaptic vesicles EM images and biochemistry suggest that a MEPP could be caused by a single vesicle EM studies revealed correlation between fusion of vesicles with plasma membrane and size of postsynaptic response

4-AP was used to vary the efficiency of release

Calcium influx is necessary for neurotransmitter release l

Voltage-gated calcium channels

Calcium influx is sufficient for neurotransmitter release

Synaptic release II The synaptic vesicle release cycle 1. Tools and Pools 2. Molecular biology and biochemistry of vesicle release: 1. Docking 2 Priming 2. 3. Fusion 3. Recovery and recycling of synaptic vesicles

The synaptic vesicle cycle

How do we study vesicle dynamics? Morphological techniques Electron microscopy to obtain static pictures of vesicle distribution; TIRFM (total internal reflection fluorescence microscopy) to visualize movement of vesicles close to the membrane

Physiological studies

Chromaffin cells Neuroendocrine cells derived from adrenal medulla with large dense-core vesicles. Can measure membrane fusion (capacitance measurements), or direct release of catecholamine transmitters using carbon fiber electrodes (amperometry) Neurons Measure release of neurotransmitter from a p presynaptic y p cell by yq quantifying y g the response of a postsynaptic cell

Ge et cs Genetics Delete or overexpress proteins in mice, worms, or flies, and analyze phenotype using the above techniques

Synaptic y p vesicle release consists of three principal steps: 1. Docking Docked vesicles lie close to plasma membrane (within 30 nm)

1. Priming Primed vesicles can be induced to fuse with the plasma membrane by sustained depolarization, high K+, elevated Ca++, hypertonic sucrose treatment

2. Fusion Vesicles fuse with the plasma membrane to release transmitter. Physiologically this occurs near calcium channels, but can be induced experimentally over larger area (see ‘priming’) priming ). The ‘active active zone zone’ is the site of physiological release, and can sometimes be recognized as an electrondense structure.

Neurotransmitter Release

Vesicle release requires many proteins on vesicle and plasma membrane p

SNAREs: targets of clostridial NTs

SNAREs: targets g of clostridial neurotoxins

Priming Vesicles in the reserve pool undergo priming to enter the readilyreleasable pool At a molecular level, priming corresponds to the assembly of the SNARE complex

The SNARE complex

Inhibitory domain, folds back on itself “open” syntaxin doesn’tt fold doesn properly

Synaptotagmin functions as a calcium sensor, promoting vesicle fusion

Calcium & exocytosis

Regulation by calcium: through synaptotagmin? t t i ?

Mutants of syt

SNARE et synaptotagmine

Syt accelerates membrane fusion in vitro

Annuall Reviews i

Syt acts through SNAREs and lipids

Regulation by complexin & synaptotagmin y p g

A complexin-tagmin cycle?

Regulation, regulation • Much more is known: Munc 13 Munc-13

Munc 18 Munc-18

• Much more to come: ?????

Synaptic vesicles exist in multiple pools within the nerve terminal

(Release stimulated by flash-photolysis of caged calcium)

(reserve pool)

B h Becherer, U U, R Rettig, tti J. J Cell C ll Tissue Ti Res R (2006) 326 326:393 393 Morphologically, vesicles are classified as docked or undocked. Docked vesicles are further subdivided into primed and unprimed pools depending g on whether + they are competent to fuse when cells are treated with high K , elevated Ca++, sustained depolarization, or hypertonic sucrose treatment.

In CNS neurons, vesicles are divided into R Reserve pooll (80 (80-95%) 95%) Recycling pool (5-20%) Readily-releasable y pool ((0.1-2%; 5-10 synapses p y p p per active zone)) Rizzoli, Betz (2005). Nature Reviews Neuroscience 6:57-69)

A small fraction of vesicles (the recycling pool) replenishes the RRP upon mild stimulation. Strong stimulation causes the reserve pool to mobilize and be released

Docking: UNC-18 (or munc-18) is necessary for vesicle docking (W i (Weimer ett al. l 2003, 2003 N Nature t N Neuroscience i 6 6:1023) 1023)

1. unc unc-18 18 mutant C. elegans have neurotransmitter release defect 2. unc-18 mutant C. elegans have reduction of docked vesicles

Unc-18 mutants are defective for evoked and spontaneous release

Unc-18 mutants are defective for calcium-independent release

primed vesicles occasionally fuse in the absence of calcium; a calcium-independent fusion defect suggests a lack of primed vesicles

UNC-18 (munc18) is required for docking: unc-18 unc 18 mutants have fewer docked vesicles

Summary: Unc-18 mutants are unable to dock vesicles efficiently. Impaired docking leads to fewer primed vesicles; fewer primed vesicles leads to reduced overall neurotransmitter release. release

Synaptic vesicles recycle post-fusion

Modern methods to track recycling membrane

Endocytosis retrieves synaptic vesicle membrane and protein from the plasma p p membrane following g fusion The ATP-ase NSF disassembles the SNARE complex